Methods and Compositions for Modulating Flowering and Maturity in Plants

ABSTRACT

The present invention provides compositions and methods for modulating flowering time in plants. Maize RAP2.7 nucleotide sequences are disclosed which upon overexpression cause later flowering and when inhibited cause earlier flowering. Also disclosed is a DNA sequence which acts as a regulator/enhancer of RAP2.7, termed VGT1. This sequence does not code for any known protein, but acts as either a RNAi element or a regulatory DNA or RNA element that either directly regulates expression of flowering genes such as Rap2.7 or specifically targets expression of other genes which control flowering genes such as Rap2.7. This element this can be used as a sequence—based marker to identify inbred and hybrids which have altered maturity. Methods for expressing these nucleotide sequences in a plant for modifying maturity and flowering in plants are provided as well as expression constructs, vectors, transformed cells and plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. application Ser. No.12/276,961, filed Nov. 24, 2008 which application claims priority ofU.S. application Ser. No. 11/190,339 filed Jul. 27, 2005, now U.S. Pat.No. 7,479,584 issued Jan. 20, 2009 and Provisional Application Ser. No.60/592,268 filed Jul. 29, 2004, all of which are hereby incorporated byreference in their entirety.

FIELD

This invention is related to compositions and methods for affectingflowering time in plants.

BACKGROUND

Plants have two basic growth modes during their life cycles—vegetativegrowth and flower and seed growth. Above ground vegetative growth of theplant develops from the apical meristem. This vegetative meristem givesrise to all of the leaves that are found on the plant. The plant willmaintain its vegetative growth pattern until the apical meristemundergoes a change. This change actually alters the identity of themeristem from a vegetative to an inflorescence meristem. Theinflorescence meristem produces small leaves before it next producesfloral meristems. It is the floral meristem from which the flowerdevelops.

From a genetic perspective, two phenotypic changes that controlvegetative and floral growth are programmed in the plant. The firstgenetic change involves the switch from the vegetative to the floralstate. If this genetic change is not functioning properly, thenflowering will not occur. The second genetic event follows thecommitment of the plant to form flowers. The observation that the organsof the plant develop in a sequential manner suggests that a geneticmechanism exists in which a series of genes are sequentially turned onand off.

Flowering time is an important agronomic trait in cultivated plantspecies as it determines in large measure the growing region ofadaptation. Most angiosperm species are induced to flower in response toenvironmental stimuli such as day length and temperature, and internalcues, such as age. Genetic analysis revealed that there are severaltypes of mutants that alter flowering time.

Studies of two distantly related dicotyledons, Arabidopsis thaliana andAntirrhinum majus, has led to the identification of three classes ofhomeotic genes, acting alone or in combination to determine floral organidentity (Bowman, et al., (1991) Development 112:1; Carpenter and Coen,(1990) Genes Devl. 4:1483; Schwarz-Sommer, et al., (1990) Science250:931). Several of these genes are transcription factors whoseconserved DNA-binding domain has been designated the MADS box(Schwarz-Sommer, et al., supra).

Earlier acting genes that control the identity of flower meristems havealso been characterized. Flower meristems are derived from inflorescencemeristems in both Arabidopsis and Antirrhinum. Two factors that controlthe development of meristematic cells into flowers are known. InArabidopsis, the factors are the products of the LEAFY gene (Weigel, etal., (1992) Cell 69:843) and the APETALA1 gene (Mandel, et al., (1992)Nature 360:273). When either of these genes is inactivated by mutation,structures combining the properties of flowers and inflorescence develop(Weigel, et al., supra; Irish and Sussex, (1990) Plant Cell 2:741). InAntirrhinum, the homologue of the Arabidopsis LEAFY gene is FLORICAULA(Coen, et al., (1990) Cell 63:1311) and that of the APETALA1 gene isSQUAMOSA (Huijser, et al., (1992) EMBO J. 11:1239). The latter paircontains MADS box domains.

Genetic studies in Arabidopsis thaliana have identified five genes(APETALA1 (AP1), APETALA2 (AP2), APETALA3 (AP3), PISTILLATA (PI) andAGAMOUS (AG)) that are involved in the specification of floral organidentity. Mutations in these genes result in homeotic transformation ofone organ type into another, much like the homeotic selector genes inanimal development. These five genes act in spatially localized domainsin a flower and in different combinations to specify the development ofthe sepals, petals, stamens and carpels. All five genes encode proteinswhich appear to function as transcription factors. Four of theseproteins are members of the MADS domain family of dimeric transcriptionfactors. MADS domain proteins are found in many organisms includingyeast, mammals, insects and plants. The fifth protein, AP2, is a memberof another class of DNA binding proteins which may be unique to plants

APETALA2 (AP2) plays an important role in the control of Arabidopsisflower and seed development and encodes a putative transcription factorthat is distinguished by a novel DNA binding motif referred to as theAP2 domain. The AP2 domain containing or RAP2 (related to AP2) family ofproteins is encoded by a minimum of 12 genes in Arabidopsis. The RAP2genes encode two classes of proteins, AP2-like and EREBP-like, that aredefined by the number of AP2 domains in each polypeptide as well as bytwo sequence motifs referred to as the YRG and RAYD elements that arelocated within each AP2 domain. RAP2 genes are differentially expressedin flower, leaf, inflorescence stem, and root. Moreover, the expressionof at least three RAP2 genes in vegetative tissues are controlled byAP2. Thus, unlike other floral homeotic genes, AP2 is active during bothreproductive and vegetative development.

Maize is a monocotyledonous plant species and belongs to the grassfamily. It is unusual for a flowering plant as it has unisexualinflorescences. The male inflorescence (tassel) develops in a terminalposition, whereas the female inflorescences (ears) grow in the axil ofvegetative leaves. The inflorescences, as typical for grasses, arecomposed of spikelets. In the case of maize each spikelet contains twoflorets (the grass flower) enclosed by a pair of bracts (inner and outerglume). A number of genes have been identified which modify floweringtime in maize including Id1 and DLF.

There is increasing incentive by those working in the field of plantbiotechnology to successfully genetically engineer plants, including themajor crop varieties. One genetic modification that would beeconomically desirable would be to accelerate the flowering time of aplant. Induction of flowering is often the limiting factor for growingcrop plants. One of the most important factors controlling induction offlowering is day length, which varies seasonally as well asgeographically. There is a need to develop a method for controlling andinducing flowering in plants, regardless of the locale or theenvironmental conditions, thereby allowing production of crops, at anygiven time. Since most crop products (e.g. seeds, grains, fruits), arederived from flowers, such a method for controlling flowering would beeconomically invaluable.

It is an object of the present invention to provide methods andcompositions for affecting flowering time in plants.

It is yet another object of the invention to provide novel nucleotidesequences isolated from maize which encode proteins which affectflowering time in plants.

It is yet another object of the invention to provide maize RAP2.7 geneswhich affect flowering time and internode length in maize.

It is yet another object of the invention to provide DNA regulatoryfactors which enhance/inhibit the ability of RAP2.7 to regulateflowering time.

It is yet another object of the invention to provide methods andcompositions including nucleotide constructs, vectors, transgenic cellsand plants with altered flowering characteristics as described herein.

It is yet another object of the invention to provide markers foridentification of mutant plants which may have altered flowering time bythe presence of marker VGT1 sequences.

SUMMARY

Compositions and methods involved in the modulation of flowering inplants are provided. The compositions include nucleic acid moleculesisolated from maize which encode RAP2.7 proteins. Amino acid sequencesof these proteins are also provided. Further, polynucleotides havingnucleic acid sequences encoding maize RAP2.7 proteins are also provided.These proteins and the nucleotide sequences encoding them provide anopportunity to manipulate maturity of plants. When polynucleotidesequences encoding the RAP2.7 gene product are overexpressed floweringtime is delayed, and when the product is inhibited flowering is earlier.

Typically maturity of a given plant is changed with crossing of earlierand later maturity plants. Agronomic traits such as yield or lodgingresistance are often lost in the process. The compositions of theinvention provide for the ability to make significant changes inmaturity while keeping other vegetative and reproductive characteristicssimilar using a transgenic approach.

The invention includes methods for manipulating the maturity of plantsusing polynucleotide sequences that were isolated from maize (Zea mays).These sequences alone, or in combination with other sequences, can beused to control plant maturity and thus area of adaptation. In anotheraspect of the present invention, nucleotide constructs such asexpression cassettes and transformation vectors comprising the isolatednucleotide sequences are disclosed. The transformation vectors can beused to transform plants and express the flower modulation control genesin the transformed cells. In this manner, the maturity of plants as wellas area of adaptation can be controlled. Transformed cells as well asregenerated transgenic plants and seeds containing and expressing theisolated polynucleotide sequences and protein products are alsoprovided.

Also according to the invention, a novel DNA sequence termed (VGT1) hasbeen identified which is a 2 kb region on maize chromosome 8L. VGT1 actsas a CIS interaction type non-coding RNA sequence for maize RAP2.7. Inmutants with early flowering, VGT1 may interact or repress theexpression level of RAP2.7 causing down regulation of RAP2.7 and earlyflowering. Thus the invention also comprises nucleotide sequencesencoding a VGT1 DNA factor which interacts either directly or indirectlywith RAP2.7 in modulating the flowering time in plants. The inventionincludes nucleotide sequences, polymorphisms, “expression-type”constructs with the VGT1 sequences operably linked to promoters regionsfor transcription of the same, transgenic cells and plants with alteredflowering time. The VGT1 sequences and the alternate forms thereof mayalso be used as markers to identify plants with flowering that may bedifferent from wild type.

For any of the sequences disclosed herein, the polynucleotide of theinvention or at least 20 contiguous bases therefrom may be used asprobes to isolate and identify similar genes in other plant species. Thesequences disclosed may also be used to isolate regulatory elements andpromoter sequences that are natively associated with the polynucleotidesdisclosed herein to give spatial and temporal expression of operativelylinked sequences to flowering in plants.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the levels of RAP2.7 expression at Day: 14,day 20 and day 27.

FIG. 2 is an illustration of the over-expression vector used intransformation, showing the portion between the right and left T-DNAborders (RB, LB). The transformation vector for RAP2.7 over-expression,PHP20922, was created by electroporating the JT vector PHP20921 intoAgrobacterium. The Invitrogen (Carlsbad, California) Gateway technologywas used to create PHP20921. Specifically, the RAP2.7 coding region wasfirst amplified by PCR with 5′-primer(ggggacaagtttgtacaaaaaagcaggctatgcagttggatctgaacgt) and 3′ primer(ggggaccactttgtacaagaaagctgggttcagcggggatggtgatg). These primers containGateway attB recombination sites. The PCR product was confirmed bysequencing and cloned into a Gateway vector pDONR221 via a BPrecombination reaction as described by the vendor (Invitrogen, Carlsbad,Calif.). This resulted in the entry clone, PHP20923. Two other entryclones, PHP20830 containing the rice actin promoter, PHP20234 containingthe pinII terminator, have been previously created using similar cloningstrategies. A destination vector PHP20909 was also created frompDESTR4-R3 vector (Invitrogen, Carlsbad, Calif.) by inserting anexpression cassette of the CaMV35S promoter driving the herbicideresistance gene Bar followed by a pinII terminator. The four vectors,PHP20923, 20830, 20234 and 20909, were then used to create the JTvector, PHP20921, via a LR recombination reaction following vendor'sinstructions (Invitrogen, Carlsbad, Calif.).

FIG. 3 is the deduced amino acid sequence of RAP2.7 from C22-4 allele.The two putative AP2 domains are highlighted in bold, whereas the linkerregion between them is italicized. The well conserved YRG and RAYDmotifs are underlined, although there is an R to K substitution in thesecond RAYD motif.

FIG. 4 is a GAP alignment between the genomic RAP2.7 from B73 comparedto the sequence of RAP2.7 of M017. Gap Weight: 50; Average Match:10.000; Length Weight: 3; Average Mismatch: 0.000; Quality: 59308;Length: 8586; Ratio: 7.730; Gaps: 41; Percent Similarity: 87.482,Percent Identity: 87.482. Vladutu, et al., (1999) Genetics 153:993-1007.

FIG. 5 is an illustration of construction of the RAP2.7 gene fragmentsfor the RNA interference vector. Fragment TR1 (Truncated1) was createdby PCR using forward (ggatccgatctgaacgtggccgag) and reverse primers(gaattcctaggcagctgttcttgtctctttg) corresponding to roughly ⅔ of thecoding sequence starting from 9 bp downstream of ATG. Fragment IR1(Inverted1) was generated similarly by PCR with forward(gcggccgcgatctgaacgtggccgag) and reverse primers(gaattctgtgggactcccagcggcctgtgc) starting from the same position as TR1,although IR1 is only half the length of TR1.

FIG. 6 is an illustration of the vector used in transformation showingonly the portion between the right and left T-DNA borders (RB, LB). Asdescribed in FIG. 5, fragments corresponding to truncated coding regionsto be used for the RNA interference construct were generated by as TR1,flanked by BamH1 and EcoR1, and IR1, flanked by Notl and EcoR1restriction sites. Vector PHP16501 containing the rice actin promoterwas linearized with Notl and BamH1. In a 3-piece ligation, TR1 wascloned in downstream of Notl followed by IR1 in reverse orientation. Theresulting cassette vector, PHP21767, was then cloned into a JT vectorcontaining the CaMV35S promoter driving the herbicide resistance geneBar. The transformation vector for RAP2.7 down regulation, PHP21842, wasgenerated by electroporating PHP21798 into Agrobacterium.

FIG. 7 is the data showing the fine genetic and physical mapping ofVGT1. First row indicates physical distance (in kb) from Rap2.7, basedon sequence derived from the relevant Mo17 BAC clone from library.Second row indicates the type of molecular marker. Third row indicatesthe name of the molecular markers. Rows from 4 to 21 indicate thegenotype of parental lines (N28 and C22-4) and of the 17 segmentalQTL-Nearly Isogenic Lines. The VGT1 column shows where the QTL wasmapped. The last two columns provide the phenotypic scores for DPS (Daysto Pollen Shed) and ND (plant node number).

FIG. 8 is a graph showing the results of association mapping for markersdeveloped at the Vgt1-Rap2.7 region and phenotypic data for floweringtime collected in Bologna (2002 and 2003) among a set of 96 maize inbredlines. Statistical association is expressed as P from ANOVA tests.

FIG. 9 is an alignment of the VGT1 sequences from all four lines,including a consensus sequence line N28 was identical to B73.

DETAILED DESCRIPTION

The present invention provides, inter alia, compositions and methods formanipulating flowering time in plants. As used herein the term“flowering time” or maturity shall mean the time at which a plantreaches physiological maturity and is capable of reproducing.

The compositions comprise nucleic acid molecules comprising sequences ofplant genes and the polypeptides encoded thereby as well as regulatoryfactors which are non-coding. Particularly, the nucleotide and aminoacid sequences for a maize RAP2.7 (related to AP2 domain containing)gene are provided. Three RAP2.7 encoding nucleotide sequences areprovided at SEQ ID NOS: 1 (cDNA), 3 (genomic) and 4 (genomic) with thecorresponding protein at SEQ ID NO: 2. Three VGT1 nucleotide sequencesare provided at SEQ ID NOS: 5, 6 and 7 with the consensus sequence atSEQ ID NO:8. As discussed in more detail below, the sequences of theinvention are involved in many basic biochemical pathways that regulateflowering time and maturity in plants. Thus, methods are provided forthe expression of these sequences in a host plant to modulate plantflowering. Some of the methods involve stably transforming a plant witha nucleotide sequence capable of modulating plant flowering operablylinked with a promoter capable of driving expression (or transcription)of a nucleotide sequence in a plant cell.

Promoter and other regulatory elements which are natively associatedwith these genes can be easily isolated using the sequences and methodsdescribed herein with no more than routine experimentation. Thesesequences can be used to identify promoter, enhancer or other signalingsignals in the regulatory regions of RAP2.7 encoding sequences. Theseregulatory and promoter elements provide for temporal and spatialexpression of operably linked sequences with flowering in a plant.Methods are provided for the regulated expression of a nucleotidesequence of interest that is operably linked to the promoter regulatorysequences disclosed herein. Nucleotide sequences operably linked to thepromoter sequences are transformed into a plant cell. Exposure of thetransformed plant to a stimulus such as the timing of flowering inducestranscriptional activation of the nucleotide sequences operably linkedto these promoter regulatory sequences.

By “heterologous nucleotide sequence” is intended a sequence that is notnaturally occurring with the referenced sequence. While the referencednucleotide sequence is heterologous to the promoter sequence or viceversa, it may be homologous, or native, or heterologous, or foreign, tothe plant host.

By “operably linked” is intended a functional linkage between a promotersequence and a second sequence, wherein the promoter sequence initiatesand mediates transcription of the DNA sequence corresponding to thesecond sequence. Generally, operably linked means that the nucleic acidsequences being linked are contiguous and, where necessary to join twoprotein coding regions, contiguous and in the same reading frame. In thecase of a DNA regulatory factor the nucleic acid sequences aretranscribed only.

A polypeptide is said to have RAP2.7-like activity when it has one ormore of the properties of the native protein. It is within the skill inthe art to assay protein activities obtained from various sources todetermine whether the properties of the proteins are the same. In sodoing, one of skill in the art may employ any of a wide array of knownassays including, for example, biochemical and/or pathological assays.For example, one of skill in the art could readily produce a planttransformed with a RAP2.7 polypeptide variant and assay a property ofnative RAP2.7 protein in that plant material to determine whether aparticular RAP2.7 property was retained by the variant.

The compositions and methods of the invention are involved inbiochemical pathways and as such may also find use in the activation ormodulation of expression of other genes, including those involved inother aspects of flowering time.

Although there is some conservation among these genes, proteins encodedby members of these gene families may contain different elements ormotifs or sequence patterns that modulate or affect the activity,subcellular localization, and/or target of the protein in which they arefound. Such elements, motifs or sequence patterns may be useful inengineering novel enzymes for modulating gene expression in particulartissues. By “modulating” or “modulation” is intended that the level ofexpression of a gene may be increased or decreased relative to genesdriven by other promoters or relative to the normal or uninduced levelof the gene in question.

According to the invention, overexpression of maize RAP2.7 causedflowering that was later than normal in plants as well as an increasednumber of leaves (nodes) produced prior to flowering resulting in tallerplant stature. Inhibition of maize RAP2.7 caused maturation or floweringthat was earlier than normal. Also the presence of mutant VGT1correlated with earlier flowering as well, thus leading to the conceptthat VGT1 acts as direct or indirect enhancer/regulator of RAP2.7.Expression of the proteins encoded by RAP2.7 encoding sequences ortranscription of the VGT1 regulatory element can be used to modulate orregulate the expression of proteins in these flowering pathways andother directly or indirectly affected pathways. Hence, the compositionsand methods of the invention find use in altering plant flowering andmaturity. In other embodiments, fragments of the sequences are used toconfer desired properties to synthetic constructs for use in regulatingplant maturity and flowering.

The present invention provides for isolated nucleic acid moleculescomprising nucleotide sequences encoding the amino acid sequence shownin SEQ ID NO: 2 as well as their conservatively modified variants.Further provided are polypeptides having an amino acid sequence encodedby a nucleic acid molecule described herein, for example thosepolypeptides comprising the sequences set forth in SEQ ID NO: 1, 3 and4, and fragments and variants thereof.

The present invention further provides for an isolated nucleic acidmolecule comprising the sequences shown in SEQ ID NO: 1, 3, 4, 5, 6, 7or 8.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In someembodiments, an “isolated” nucleic acid is free of sequences (such asother protein-encoding sequences) that naturally flank the nucleic acid(i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) inthe genomic DNA of the organism from which the nucleic acid is derived.For example, in various embodiments, the isolated nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.4kb, 0.3 kb, 0.2 kb, or 0.1 kb, or 50, 40, 30, 20 or 10 nucleotides thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. A protein that is substantiallyfree of cellular material includes preparations of protein having lessthan about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, culture medium may represent less than about30%, 20%, 10% or 5% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences whichretain the functional properties of the encoded peptide or of thenon-coding RNA are encompassed by the present invention. By “fragment”is intended a portion of the nucleotide sequence or a portion of theamino acid sequence and hence protein encoded thereby. Fragments of anucleotide sequence may encode protein fragments that retain thebiological activity of the native protein and hence affect flowering byretaining RAP2.7-like activity or may include portions of non-codingregulatory element which retain the RAP2.7 modulating activity of VGT1.Alternatively, fragments of a nucleotide sequence that are useful ashybridization probes generally do not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides and up to the full-length nucleotide sequence encoding theproteins or regulating RAP2.7 of the invention.

A fragment of a RAP2.7 nucleotide sequence that encodes a biologicallyactive portion of a RAP2.7 protein of the invention will encode at least12, 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425 or 450, contiguous amino acids, or up to the totalnumber of amino acids present in a full-length RAP2.7 protein of theinvention.

A fragment of a VGT1 nucleotide sequence that encodes a biologicallyactive non-transcribed RNA of the invention will encode at least 12, 25,30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675 or 680contiguous nucleotide bases or up to the total number of nucleotidespresent in a full-length VGT1 regulatory element of the invention.

Fragments of a RAP2.7 or VGT1 nucleotide sequence that are useful ashybridization probes or PCR primers generally need not encode abiologically active portion of a protein or RNA. Thus, a fragment of aRAP2.7 or VGT1 nucleotide sequence may encode a biologically activeportion of a RAP2.7 protein or a biologically active non-coding RNA, orit may be a fragment that can be used as a hybridization probe or PCRprimer using methods disclosed below. A biologically active portion of aRAP2.7 protein or VGT1 regulatory element can be prepared by isolating aportion of the RAP2.7 or VGT1 nucleotide sequences of the invention,expressing the encoded portion of the Rap2.7 or the active portion ofthe VGT1 regulatory element (e.g., by recombinant expression in vitro),and assessing the activity of the encoded portion of the RAP2.7 proteinor the regulating ability of the regulatory element on RAP2.7. Nucleicacid molecules that are fragments of a RAP2.7 or VGT1 nucleotidesequence comprise at least 16, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300 or 2400 nucleotides, orup to the number of nucleotides present in a full RAP2.7 or VGT1nucleotide sequence disclosed herein.

By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the polypeptides of the invention. Naturallyoccurring allelic variants such as these can be identified with the useof well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically-derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a RAP2.7 protein orVGT1 regulatory element of the invention. Generally, variants of aparticular nucleotide sequence of the invention will have at least 40%,50%, 60%, 70%, generally at least 75%, 80%, 85%, or about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular nucleotide sequence as determined by sequence alignmentprograms described elsewhere herein using default parameters.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess the desired biological activity of the native protein, hencethey will continue to possess at least one activity possessed by thenative RAP2.7 protein. Such variants may result from, for example,genetic polymorphism or from human manipulation. Biologically activevariants of a RAP2.7 native protein of the invention will have at least40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, 86%, 87%, 88%,89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to the amino acid sequence for the native protein asdetermined by sequence alignment programs described elsewhere hereinusing default parameters. A biologically active variant of a protein ofthe invention may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, as few as 5, as few as 4, 3, 2 or even 1 aminoacid residue. As used herein, reference to a particular nucleotide oramino acid sequence (a RAP2.7 or VGT1 sequence) shall include allmodified variants as described supra.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of RAP2.7 proteins can be preparedby mutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel, (1985)Proc. Nad. Acad. Sci. USA 82:488-492; Kunkel, et al., (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff, et al., (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be made.

Thus, the nucleotide sequences of the invention include both naturallyoccurring sequences as well as mutant forms. Likewise, the proteins ofthe invention encompass both naturally-occurring proteins as well asvariations and modified forms thereof. Such variants whether protein ornucleotide will continue to possess the desired RAP2.7 or VGT1-likeactivity. It is recognized that variants need not retain all of theactivities and/or properties of the native RAP2.7 or VGT1. Obviously,the mutations that will be made in the DNA encoding the RPA2.7 variantmust not place the sequence out of reading frame and in some embodimentswill not create complementary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication Number 75,444.

The deletions, insertions and substitutions of the protein or nucleotidesequences encompassed herein are not expected to produce radical changesin the characteristics of the RAP 2.7 protein or VGT1 regulatoryelement. However, when it is difficult to predict the exact effect ofthe substitution, deletion or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays. That is, the activity of RAP2.7 polypeptidesor VGT1 can be evaluated by either a change in flowering time ormaturity when the encoded protein or regulatory element is altered.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different RAP2.7 orVGT1 coding sequences can be manipulated to create a new RAP2.7 or VGT1possessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the RAP2.7encoding polynucleotide of the invention and other known flowering genesto obtain a new gene coding for a protein with an improved property ofinterest, such as an increased K_(m) in the case of an enzyme.Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer, (1994) Nature 370:389391; Crameri, et al., (1997) NatureBiotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347;Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri,et al., (1998) Nature 391:288-291 and U.S. Pat. Nos. 5,605,793 and5,837,458.

The compositions of the invention also include isolated nucleic acidmolecules comprising the promoter nucleotide sequences nativelyassociated with the RAP2.7 polynucleotides. By “promoter” is intended aregulatory region of DNA usually comprising a TATA box capable ofdirecting RNA polymerase II to initiate RNA synthesis at the appropriatetranscription initiation site for a particular coding sequence. Apromoter may additionally comprise other recognition sequences generallypositioned upstream or 5′ to the TATA box, referred to as upstreampromoter elements, which influence the transcription initiation rate.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other crop plants. In this manner, methods such asPCR, hybridization, and the like can be used to identify such sequencesbased on their sequence homology to the sequences set forth herein.Sequences isolated based on their sequence identity to the nucleotidesequences set forth herein or to fragments thereof are encompassed bythe present invention. Such sequences include sequences that areorthologs of the disclosed sequences. By “orthologs” is intended genesderived from a common ancestral gene and which are found in differentspecies as a result of speciation. Genes found in different species areconsidered orthologs when their nucleotide sequences and/or theirencoded protein sequences share substantial identity as definedelsewhere herein. Functions of orthologs are often highly conservedamong species. Thus, isolated sequences that have RAP2.7 or VGT1activity or encode a RAP2.7 protein and which hybridize under stringentconditions to RAP2.7 or VGT1 sequences disclosed herein, or to fragmentsthereof, are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press Plainview, N.Y.).See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methodsand Applications (Academic Press, New York); Innis and Gelfand, eds.(1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand,eds. (1999) PCR Methods Manual (Academic Press, New York). Known methodsof PCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present it a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments or other oligonucleotides and may belabeled with a detectable group such as ³²P or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the disease-resistantsequences of the invention. Methods for preparation of probes forhybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook, et al., (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

For example, an entire sequence disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding flowering or maturity regulating sequences,including promoters and messenger RNAs. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique among flowering or maturity related sequencesand may be at least about 10 or 15 or 17 nucleotides in length or atleast about 20 or 22 or 25 nucleotides in length. Such probes may beused to amplify corresponding sequences from a chosen organism by PCR.This technique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook, et al., (1989) Molecular Cloning:A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different under differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length or lessthan 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3. Incubationshould be at a temperature of least about 30° C. for short probes (e.g.,10 to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide. Exemplarylow stringency conditions include hybridization with a buffer solutionof 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl, 0.3 M trisodiumcitrate) at 50 to 55° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. for 20 minutes.Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours.

Specificity is typically a function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m), can be approximated fromthe equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284:Trn=81.5° C.+16.6 (log M)+0.41 (% GC)-−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m), is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m), is reduced by about 1°C. for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m), can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence and its complement at a defined ionic strength andpH. However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3 or 4° C. lower than the thermal melting point(T_(m)); moderately stringent conditions can utilize a hybridizationand/or wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting point(Tm); low stringency conditions can utilize a hybridization and/or washat 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point(Tm). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m), ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), theSSC concentration may be increased so that a higher temperature can beused. An extensive guide to the hybridization of nucleic acids is foundin Tijssen, (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

In general, sequences that encode a RAP2.7 protein or which encode aVGT1 regulatory element and which hybridize to the RAP2.7 or VGT1sequences disclosed herein will be at least about 70% homologous, andeven about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or morehomologous with the disclosed sequences. That is, the sequence identityof the sequences may be from about 70% or 75%, and even about 80%, 85%,87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical orhigher, so that the sequences may differ by only 10, 9, 8, 7, 6, 5, 4,3, 2 or 1 amino acid residue or by 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleic acid.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity” and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100 or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, (1988) CABIOS 4:11-17; the local homology algorithmof Smith, et al., (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman, (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul, (1990)Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul,(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins, et al.,(1988) Gene 73:237-244; Higgins, et al., (1989) CABIOS 5:151-153;Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al.,(1992) CABIOS 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-331. The ALIGN program is based on the algorithm of Myers andMiller, (1988) supra. A PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4 can be used with the ALIGN programwhen comparing amino acid sequences. The BLAST programs of Altschul, etal., (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlinand Altschul, (1990) supra. BLAST nucleotide searches can be performedwith the BLASTN program, score=100, wordlength=12, to obtain nucleotidesequences homologous to a nucleotide sequence encoding a protein of theinvention. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul, et al., (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul, et al., (1997) supra. When utilizing BLAST,Gapped BLAST or PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used (see information at www.ncbi.nlm.nih.gov). Alignment mayalso be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP version 10 using thefollowing parameters: % identity using GAP Weight of 50 and LengthWeight of 3; % similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10. GAP uses thealgorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-453, tofind the alignment of two complete sequences that maximizes the numberof matches and minimizes the number of gaps. GAP considers all possiblealignments and gap positions and creates the alignment with the largestnumber of matched bases and the fewest gaps. It allows for the provisionof a gap creation penalty and a gap extension penalty in units ofmatched bases. GAP must make a profit of gap creation penalty number ofmatches for each gap it inserts. If a gap extension penalty greater thanzero is chosen, GAP must, in addition, make a profit for each gapinserted of the length of the gap times the gap extension penalty.Default gap creation penalty values and gap extension penalty values inVersion 10 of the Wisconsin Genetics Software Package for proteinsequences are 8 and 2, respectively. For nucleotide sequences thedefault gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the Wisconsin Genetics Software Package isBLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 80%,85%, 90%, 95% or higher sequence identity compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or at least 95% or higher sequenceidentity.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Trr,) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C. lower than theT_(m), depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologicallycross-reactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 75%,80%, 83%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98% or 99% or highersequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch, (1970) J Mol. Biol.48:443453. An indication that two peptide sequences are substantiallyidentical is that one peptide is immunologically reactive withantibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. Peptides that are“substantially similar” share sequences as noted above except thatresidue positions that are not identical may differ by conservativeamino acid changes.

Methods for altering flowering time in a plant are provided. In someembodiments, the methods involve stably transforming a plant with a DNAconstruct comprising a nucleotide sequence of the invention operablylinked to a promoter that drives expression (or transcription) in aplant. While the choice of promoter will depend on the desired timingand location of expression of the nucleotide sequences, desirablepromoters include constitutive and tissue specific promoters. Thesemethods may find use in agriculture, particularly in changing thematurity of a particular crop plant to alter its area of adaptation.Thus, transformed plants, plant cells, plant tissues and seeds thereofare provided by the present invention.

In another embodiment, the methods of the present invention involveidentifying phenotypes associated with an altered flowering time by lossof RAP2.7 or VGT1 activity in plants that contain transposon insertionswithin the nucleotide sequences herein.

In some embodiments, the nucleic acid molecules comprising RAP2.7 orVGT1 sequences of the invention are provided in expression cassettes ornucleotide constructs for expression/transcription in the plant ofinterest. Such cassettes will include 5′ and 3′ regulatory sequencesoperably linked to a RAP2.7 or VGT1 nucleotide sequence of theinvention. By “operably linked” is intended a functional linkage betweena promoter and a second sequence, wherein the promoter sequenceinitiates and mediates transcription of the DNA sequence correspondingto the second sequence. The cassette may additionally contain at leastone additional nucleotide sequence to be cotransformed into theorganism. Alternatively, the additional sequence(s) can be provided onmultiple expression cassettes or nucleotide construct.

Such an expression cassette or nucleotide construct is provided with aplurality of restriction sites for insertion of the RAP2.7 or VGT1sequence to be under the transcriptional regulation of the regulatoryregions. The expression cassette or nucleotide construct mayadditionally contain selectable marker genes. The expression cassettewill include in the 5′-3′ direction of transcription, a transcriptionaland if necessary a translational initiation region, a RAP2.7 or VGT1nucleotide sequence of the invention, and a transcriptional and ifnecessary, translational termination region functional in plants. Thetranscriptional initiation region, or promoter, may be native oranalogous or foreign or heterologous to the plant host. Additionally,the promoter may be the natural sequence or alternatively a syntheticsequence. By “foreign” is intended that the transcriptional initiationregion is not found in the native plant into which the transcriptionalinitiation region is introduced. As used herein, a “chimeric gene”comprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

While it may be preferable to regulate RAP2.7 or VGT1 sequences usingheterologous promoters, the native promoter sequences may be used. Suchconstructs would change expression levels of the RAP2.7 or amounts ofthe VGT1 regulatory element present in the plant or plant cell. Thus,the phenotype of the plant or plant cell is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interestor may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See also,Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot,(1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149;Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990)Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639. Whereappropriate, the gene(s) may be optimized for increased expression inthe transformed plant. That is, the genes can be synthesized usingplant-preferred codons for improved expression. Methods are available inthe art for synthesizing plant-preferred genes. See, for example, U.S.Pat. Nos. 5,380,831 and 5,436,391 and Murray, et al., (1989) NucleicAcids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats and other such well characterized sequences thatmay be deleterious to gene expression. The G-C content of the sequencemay be adjusted to enhance expression in a given host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes/nucleotide constructs may additionally contain5′ leader sequences in the expression cassette construct. Such leadersequences can act to enhance translation. Translation leaders are knownin the art and include: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al., (1989)Proc. Nat'l. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Allison, et al., (1986); MDMVleader (Maize Dwarf Mosaic Virus); Virology 154:9-20) and humanimmunoglobulin heavy-chain binding protein (BiP), (Macejak, et al.,(1991) Nature 353:90-94); untranslated leader from the coat protein mRNAof alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature325:622625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989)in Molecular Biology of RNA, ed. Cech, (Liss, New York), pp. 237-256)and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991)Virology 81:382-385). See also, Della-Cioppa, et al., (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In those instances where it is desirable to have the expressed productof the heterologous nucleotide sequence of interest directed to aparticular organelle, such as the chloroplast or mitochondrion, orsecreted at the cell's surface or extracellularly, the expressioncassette may further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to, the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase, and the like.

In preparing the expression cassette/nucleotide construct, the variousDNA fragments may be manipulated so as to provide for the DNA sequencesin the proper orientation and, as appropriate, in the proper readingframe. Toward this end, adapters or linkers may be employed to join theDNA fragments or other manipulations may be involved to provide forconvenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g.,transitions and transversions, may be involved.

Generally, the expression cassette/nucleotide construct will comprise aselectable marker gene for the selection of transformed cells.Selectable marker genes are utilized for the selection of transformedcells or tissues. Marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II (NEO)and hygromycin phosphotransferase (HPT), as well as genes conferringresistance to herbicidal compounds, such as glufosinate ammonium,bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Seegenerally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511;Christopherson, et al., (1992) Proc. Nad. Acad. Sci. USA 89:6314-6318;Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol.6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, etal., (1987) Cell 48:555-566; Brown, et al., (1987) Cell 49:603-612;Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc.Nad. Acad. Sci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Nad.Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science248:480-483; Gossen, (1993) Ph.D. Thesis, University of Heidelberg;Reines, et al., (1993) Proc. Nad. Acad. Sci. USA 90:1917-1921; Labow, etal., (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti, et al., (1992)Proc. Nad. Acad. Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Nad.Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104;Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al.,(1992) Proc. Nad. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985)Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag,Berlin); Gill, et al., (1988) Nature 334:721-724. Such disclosures areherein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention. Anumber of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acids can be combined with constitutive, tissue-preferred, orother promoters for expression in plants. Constitutive promotersinclude, for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 1999/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171);ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723 -2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142 and 6,177,611.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to: the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners; the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides; andthe tobacco PR-1 a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters. See, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Nad. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced geneexpression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto, et al., (1997) Plant J 12(2):255-265; Kawamata, etal., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997)Mol. Gen. Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res.6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341;Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, etal., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994)Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results Probl. CellDiffer. 20:181-196; Orozco, et al., (1993) Plant Mol. Biol.23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression. Leaf-specific promoters are known in the art. See, forexample, Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al.,(1994) Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778; Gotor, et al., (1993) Plant J 3:509-18; Orozco,et al., (1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al.,(1993) Proc. Nad. Acad. Sci. USA 90(20):9586-9590.

Where low level expression is desired, weak promoters will be used.Generally, by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By low level is intendedat levels of about 1/1000 transcripts to about 1/100,000 transcripts toabout 1/500,000 transcripts per cell. Alternatively, it is recognizedthat weak promoters also include promoters that are expressed in only afew cells and not in others to give a total low level of expression.Where a promoter is expressed at unacceptably high levels, portions ofthe promoter sequence can be deleted or modified to decrease expressionlevels. Such weak constitutive promoters include, for example, the corepromoter of the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No.6,072,050), the core 35S CaMV promoter, and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463 and 5,608,142. Seealso, U.S. Pat. No. 6,177,611, herein incorporated by reference.

As used herein, “vector” refers to a molecule such as a plasmid, cosmidor bacterial phage for introducing a nucleotide construct and/orexpression cassette into a host cell. Cloning vectors typically containone or a small number of restriction endonuclease recognition sites atwhich foreign nucleotide sequences can be inserted in a determinablefashion without loss of essential biological function of the vector, aswell as a marker gene that is suitable for use in the identification andselection of cells transformed with the cloning vector. Marker genestypically include genes that provide tetracycline resistance, hygromycinresistance or ampicillin resistance.

The methods of the invention involve introducing a nucleotide constructinto a plant. By “introducing” is intended presenting to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notdepend on a particular method for introducing a nucleotide construct toa plant, only that the nucleotide construct gains access to the interiorof at least one cell of the plant. Methods for introducing nucleotideconstructs into plants are known in the art including, but not limitedto, stable transformation methods, transient transformation methods andvirus-mediated methods.

By “stable transformation” is intended that the nucleotide constructintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. By “transienttransformation” is intended that a nucleotide construct introduced intoa plant does not integrate into the genome of the plant.

The nucleotide constructs of the invention may be introduced into plantsby contacting plants with a virus or viral nucleic acids. Generally,such methods involve incorporating a nucleotide construct of theinvention within a viral DNA or RNA molecule. It is recognized that theRAP2.7 protein of the invention may be initially synthesized as part ofa viral polyprotein, which later may be processed by proteolysis in vivoor in vitro to produce the desired recombinant protein. Further, it isrecognized that promoters of the invention also encompass promotersutilized for transcription by viral RNA polymerases. Methods forintroducing nucleotide constructs into plants and expressing a proteinencoded therein, involving viral DNA or RNA molecules, are known in theart. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367 and 5,316,931, herein incorporated by reference.

A variety of other transformation protocols are contemplated in thepresent invention. Transformation protocols as well as protocols forintroducing nucleotide sequences into plants may vary depending on thetype of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing nucleotide sequencesinto plant cells and subsequent insertion into the plant genome includemicroinjection (Crossway, et al., (1986) Biotechniques 4:320-334),electroporation (Riggs, et al., (1986) Proc. Nad. Acad. Sci. USA83:5602-5606, Agrobacterium-mediated transformation (Townsend, et al.,U.S. Pat. No. 5,563,055; Zhao, et al., U.S. Pat. No. 5,981,840), directgene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, Sanford, et al., U.S.Pat. No. 4,945,050; Tomes, et al., U.S. Pat. No. 5,879,918; Tomes, etal., U.S. Pat. No. 5,886,244; Bidney, et al., U.S. Pat. No. 5,932,782;Tomes, et al., (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ CultureFundamental Methods, eds. Gamborg and Phillips (Springer-Verlag,Berlin); McCabe, et al., (1988) Biotechnology 6:923-926); and Lecltransformation (WO 2000/28058, published May 18, 2000). Also see,Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christoul, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Nad. Acad. Sci. USA85:43054309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos.5,322,783 and 5,324,646; Tomes, et al., (1995) ‘Direct DNA Transfer intoIntact Plant Cells via Microprojectile Bombardment,” in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg(Springer-Verlag, Berlin) (maize); Klein, et al., (1988) Plant Physiol.91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London)311:763-764; Bowen, et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier,et al., (1987) Proc. Nad. Acad. Sci. USA 84:5345-5349 (Liliaceae); DeWet, et al., (1985) in The Experimental Manipulation of Ovule Tissues,ed. Chapman, et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler, etal., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992)Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation);D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li,et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford,(1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) NatureBiotechnology 14:745-750 (maize via Agrobacterium tumefaciens), all ofwhich are herein incorporated by reference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure constitutive expression of the desired phenotypiccharacteristic has been achieved.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn (Zeamays), Brassica spp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.) and members of the genus Cucumis such ascucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima) and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea) and cedarssuch as Western red cedar (Thujaplicata) and Alaska yellow cedar(Chamaecyparis nootkatensis). Plants of the present invention may becrop plants (for example, alfalfa, sunflower, Brassica, cotton,safflower, peanut, sorghum, wheat, millet, tobacco, etc.), corn orsoybean plants.

Plants of particular interest include grain plants that provide seeds ofinterest, oil-seed plants and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

It is recognized that with these nucleotide sequences, gene silencingsuch as antisense constructions complementary to at least a portion ofthe messenger RNA (mRNA) for RAP2.7 or VGT1 sequences can beconstructed. Antisense nucleotides are constructed to hybridize with thecorresponding mRNA. Modifications of the antisense sequences may be madeas long as the sequences hybridize to and interfere with expression ofthe corresponding mRNA. In this manner, antisense constructions having70%, 80%, 85%, 90%, 95% or more sequence identity to the correspondingantisense sequences may be used. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides or greater may be used.

Gene silencing refers to posttranscriptional interference with geneexpression. Techniques such as antisense, co-suppression, and RNAinterference (RNAi), for example, have been shown to be effective ingene silencing. (For reviews, see, Arndt and Rank, (1997) Genome40(6):785-797; Turner and Schuch, (2000) Journal of Chemical Technologyand Biotechnology 75(10):869-882; Klink and Wolniak, (2000) Journal ofPlant Growth Regulation 19(4):371-384)

Antisense technology can be used to control gene expression throughantisense DNA or RNA or through double- or triple-helix formation.Antisense techniques are discussed, for example, in Okano, (1991)Neurochem 56:560; OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee, et al., (1979) Nucleic Acids Research6:3073; Cooney, et al., (1988) Science 241:456 and Dervan, et al.,(1991) Science 251:1360. The methods are based on binding of apolynucleotide to a complementary DNA or RNA. For example, the 5′ codingportion of a polynucleotide that encodes the mature polypeptide of thepresent invention may be used to design an antisense RNA oligonucleotideof from about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription, thereby preventing transcription and the production ofcytokinin biosynthetic enzymes. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into cytokinin biosynthetic enzymes. The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of cytokininbiosynthetic enzymes. The DNAs of this invention may also be employed toco-suppress or silence the cytokinin metabolic enzyme genes; forexample, as described in PCT Patent Application Publication WO1998/36083.

The RAP 2.7 nucleotide sequence operably linked to the regulatoryelements herein can be an antisense sequence for a targeted gene. By“antisense DNA nucleotide sequence” is intended a sequence that is ininverse orientation to the 5′-to-3′ normal orientation of thatnucleotide sequence. When delivered into a plant cell, expression of theantisense DNA sequence prevents normal expression of the DNA nucleotidesequence for the targeted gene. The antisense nucleotide sequenceencodes an RNA transcript that is complementary to and capable ofhybridizing with the endogenous messenger RNA (mRNA) produced bytranscription of the DNA nucleotide sequence for the targeted gene. Inthis case, production of the native protein encoded by the targeted geneis inhibited to achieve a desired phenotypic response. Thus theregulatory sequences disclosed herein can be operably linked toantisense DNA sequences to reduce or inhibit expression of a nativeprotein in the plant seed.

It is also recognized that the level and/or activity of the polypeptidemay be modulated by employing a polynucleotide that is not capable ofdirecting, in a transformed plant, the expression of a protein or anRNA. For example, the polynucleotides of the invention may be used todesign polynucleotide constructs that can be employed in methods foraltering or mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, allof which are herein incorporated by reference. See also, WO 1998/49350,WO 1999/07865, WO 1999/25821 and Beetham, et al., (1999) Proc. Natl.Acad. Sci. USA 96:8774-8778; herein incorporated by reference. It istherefore recognized that methods of the present invention do not dependon the incorporation of the entire polynucleotide into the genome, onlythat the plant or cell thereof is altered as a result of theintroduction of the polynucleotide into a cell. In one embodiment of theinvention, the genome may be altered following the introduction of thepolynucleotide into a cell. For example, the polynucleotide, or any partthereof, may incorporate into the genome of the plant. Alterations tothe genome of the present invention include, but are not limited to,additions, deletions, and substitutions of nucleotides into the genome.While the methods of the present invention do not depend on additions,deletions, and substitutions of any particular number of nucleotides, itis recognized that such additions, deletions or substitutions comprisesat least one nucleotide.

Methods are provided to reduce or eliminate the activity of a RAP2.7polypeptide of the invention by transforming a plant cell with anexpression cassette that expresses a polynucleotide that inhibits theexpression of the RAP2.7 polypeptide. The polynucleotide may inhibit theexpression of the Rap2.7 polypeptide directly, by preventing translationof the RAP2.7 messenger RNA, or indirectly, by encoding a polypeptidethat inhibits the transcription or translation of a RAP2.7 gene encodinga RAP2.7 polypeptide. Methods for inhibiting or eliminating theexpression of a gene in a plant are well known in the art, and any suchmethod may be used in the present invention to inhibit the expression ofa RAP2.7 polypeptide.

In some embodiments of the present invention, a plant is transformedwith an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of a RAP2.7 polypeptide ofthe invention. The term “expression” as used herein refers to thebiosynthesis of a gene product, including the transcription and/ortranslation of said gene product. For example, for the purposes of thepresent invention, an expression cassette capable of expressing apolynucleotide that inhibits the expression of at least one RAP2.7polypeptide is an expression cassette capable of producing an RNAmolecule that inhibits the transcription and/or translation of at leastone RAP2.7 polypeptide of the invention. The “expression” or“production” of a protein or polypeptide from a DNA molecule refers tothe transcription and translation of the coding sequence to produce theprotein or polypeptide, while the “expression” or “production” of aprotein or polypeptide from an RNA molecule refers to the translation ofthe RNA coding sequence to produce the protein or polypeptide.

Examples of polynucleotides that inhibit the expression of a RAP2.7polypeptide are given below.

i. Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression of aRAP2.7 polypeptide may be obtained by sense suppression orcosuppression. For cosuppression, an expression cassette is designed toexpress an RNA molecule corresponding to all or part of a messenger RNAencoding a RAP2.7 polypeptide in the “sense” orientation. Overexpression of the RNA molecule can result in reduced expression of thenative gene. Accordingly, multiple plant lines transformed with thecosuppression expression cassette are screened to identify those thatshow the greatest inhibition of RAP2.7 polypeptide expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding the RAP2.7 polypeptide, all or part of the 5′and/or 3′ untranslated region of a RAP2.7 polypeptide transcript, or allor part of both the coding sequence and the untranslated regions of atranscript encoding a RAP2.7 polypeptide. In some embodiments where thepolynucleotide comprises all or part of the coding region for the RAP2.7polypeptide, the expression cassette is designed to eliminate the startcodon of the polynucleotide so that no protein product will betranslated.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin, et al., (2002) PlantCell 14:1417-1432. Cosuppression may also be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit theexpression of endogenous genes in plants are described in Flavell, etal., (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen, et al.,(1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington, (2001)Plant Physiol. 126:930-938; Broin, et al., (2002) Plant Cell14:1417-1432; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731;Yu, et al., (2003) Phytochemistry 63:753-763 and U.S. Pat. Nos.5,034,323, 5,283,184 and 5,942,657, each of which is herein incorporatedby reference. The efficiency of cosuppression may be increased byincluding a poly-dT region in the expression cassette at a position 3′to the sense sequence and 5′ of the polyadenylation signal. See, USPatent Publication Number 2002/0048814, herein incorporated byreference. Typically, such a nucleotide sequence has substantialsequence identity to the sequence of the transcript of the endogenousgene, optimally greater than about 65% sequence identity, more optimallygreater than about 85% sequence identity, most optimally greater thanabout 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and5,034,323, herein incorporated by reference.

ii. Antisense Suppression

In some embodiments of the invention, inhibition of the expression ofthe RAP2.7 polypeptide may be obtained by antisense suppression. Forantisense suppression, the expression cassette is designed to express anRNA molecule complementary to all or part of a messenger RNA encodingthe RAP2.7 polypeptide. Over expression of the antisense RNA moleculecan result in reduced expression of the native gene. Accordingly,multiple plant lines transformed with the antisense suppressionexpression cassette are screened to identify those that show thegreatest inhibition of RAP2.7 polypeptide expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the RAP2.7polypeptide, all or part of the complement of the 5′ and/or 3′untranslated region of the RAP2.7 transcript, or all or part of thecomplement of both the coding sequence and the untranslated regions of atranscript encoding the RAP2.7 polypeptide. In addition, the antisensepolynucleotide may be fully complementary (i.e., 100% identical to thecomplement of the target sequence) or partially complementary (i.e.,less than 100% identical to the complement of the target sequence) tothe target sequence. Antisense suppression may be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, 300, 400, 450, 500, 550 or greater may be used. Methods forusing antisense suppression to inhibit the expression of endogenousgenes in plants are described, for example, in Liu, et al., (2002) PlantPhysiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, eachof which is herein incorporated by reference. Efficiency of antisensesuppression may be increased by including a poly-dT region in theexpression cassette at a position 3′ to the antisense sequence and 5′ ofthe polyadenylation signal. See, US Patent Publication Number2002/0048814, herein incorporated by reference.

iii. Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of aRAP2.7 polypeptide may be obtained by double-stranded RNA (dsRNA)interference. For dsRNA interference, a sense RNA molecule like thatdescribed above for cosuppression and an antisense RNA molecule that isfully or partially complementary to the sense RNA molecule are expressedin the same cell, resulting in inhibition of the expression of thecorresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe greatest inhibition of RAP2.7 polypeptide expression. Methods forusing dsRNA interference to inhibit the expression of endogenous plantgenes are described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci.USA 95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743 andWO 1999/49029, WO 1999/53050, WO 1999/61631 and WO 2000/49035, each ofwhich is herein incorporated by reference.

iv. Hairpin RNA Interference and Intron-Containing Hairpin RNAInterference

In some embodiments of the invention, inhibition of the expression ofone or a RAP2.7 polypeptide may be obtained by hairpin RNA (hpRNA)interference or intron-containing hairpin RNA (ihpRNA) interference.These methods are highly efficient at inhibiting the expression ofendogenous genes. See, Waterhouse and Helliwell, (2003) Nat. Rev. Genet.4:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene whoseexpression is to be inhibited, and an antisense sequence that is fullyor partially complementary to the sense sequence. Thus, the base-pairedstem region of the molecule generally determines the specificity of theRNA interference. hpRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants. See, for example, Chuangand Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731 and Waterhouseand Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNAinterference to inhibit or silence the expression of genes aredescribed, for example, in Chuang and Meyerowitz, (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet.4:29-38; Pandolfini, et al., BMC Biotechnology 3:7 and US PatentApplication Publication Number 2003/0175965, each of which is hereinincorporated by reference. A transient assay for the efficiency of hpRNAconstructs to silence gene expression in vivo has been described byPanstruga, et al., (2003) Mol. Biol. Rep. 30:135-140, hereinincorporated by reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith, et al., (2000) Nature407:319-320. In fact, Smith, et al., show 100% suppression of endogenousgene expression using ihpRNA-mediated interference. Methods for usingihpRNA interference to inhibit the expression of endogenous plant genesare described, for example, in Smith, et al., (2000) Nature 407:319-320;Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods 30:289-295and US Patent Application Publication Number 2003/0180945, each of whichis herein incorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 2002/00904, herein incorporated byreference.

v. Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for the RAP2.7 polypeptide). Methodsof using amplicons to inhibit the expression of endogenous plant genesare described, for example, in Angell and Baulcombe, (1997) EMBO J.16:3675-3684, Angell and Baulcombe, (1999) Plant J. 20:357-362 and U.S.Pat. No. 6,646,805, each of which is herein incorporated by reference.

vi. Ribozymes

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of the RAP2.7 polypeptide. Thus, thepolynucleotide causes the degradation of the endogenous messenger RNA,resulting in reduced expression of the RAP2.7 polypeptide.

This method is described, for example, in U.S. Pat. No. 4,987,071,herein incorporated by reference.

vii. Small Interfering RNA or Micro RNA

In some embodiments of the invention, inhibition of the expression of aRAP2.7 polypeptide may be obtained by RNA interference by expression ofa gene encoding a micro RNA (miRNA). miRNAs are regulatory agentsconsisting of about 22 ribonucleotides. miRNA are highly efficient atinhibiting the expression of endogenous genes. See, for example Javier,et al., (2003) Nature 425:257-263, herein incorporated by reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of RAP2.7 expression, the22-nucleotide sequence is selected from a RAP2.7 transcript sequence andcontains 22 nucleotides of said RAP2.7 sequence in sense orientation and21 nucleotides of a corresponding antisense sequence that iscomplementary to the sense sequence. miRNA molecules are highlyefficient at inhibiting the expression of endogenous genes, and the RNAinterference they induce is inherited by subsequent generations ofplants.

The availability of reverse genetics systems, which are well-known inthe art, makes the generation and isolation of down-regulated or nullmutants feasible, given the availability of a defined nucleic acidsequence, as provided herein. One such system (the Trait Utility Systemfor Corn, i.e., TUSC) is based on successful systems from otherorganisms (Ballinger, et al., (1989) Proc. Natl. Acad. Sci. USA86:9402-9406; Kaiser, et al., (1990) Proc. Natl. Acad. Sci. USA87:1686-1690 and Rushforth, et al., (1993) Mol. Cell. Biol. 13:902-910).The central feature of the system is to identify Mu transposoninsertions within a DNA sequence of interest in anticipation that atleast some of these insertion alleles will be mutants. See, U.S. Pat.Nos. 6,300,542 and 5,962,764. To develop the system, DNA was collectedfrom a large population of Mutator transposon stocks that were thenself-pollinated to produced F2 seed. To find Mu transposon insertionswithin a specific DNA sequence, the collection of DNA samples isscreened via PCR using a gene-specific primer and a primer that annealsto the inverted repeats of Mu transposons. A PCR product is expectedonly when the template DNA comes from a plant that contains a Mutransposon insertion within the target gene. Once such a DNA sample isidentified, F2 seed from the corresponding plant is screened for atransposon insertion allele. Transposon insertion mutations of the an1gene have been obtained via the TUSC procedure (Bensen, et al., (1995)).This system is applicable to other plant species, at times modified inaccordance with knowledge and skills reasonably attributed to ordinaryartisans.

The use of the term “nucleotide constructs” herein is not intended tolimit the present invention to nucleotide constructs comprising DNA.Those of ordinary skill in the art will recognize that nucleotideconstructs, particularly polynucleotides and oligonucleotides, comprisedof ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. Thus, the nucleotide constructs of the present inventionencompass all nucleotide constructs that can be employed in the methodsof the present invention for transforming plants including, but notlimited to, those comprised of deoxyribonucleotides, ribonucleotides andcombinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs of the invention also encompass all forms ofnucleotide constructs including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures, andthe like.

Furthermore, it is recognized that the methods of the invention mayemploy a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or at leastone RNA, such as, for example, an antisense RNA that is complementary toat least a portion of an mRNA. Typically such a nucleotide construct iscomprised of a coding sequence for a protein or an RNA operably linkedto 5′ and 3′ transcriptional regulatory regions. Alternatively, it isalso recognized that the methods of the invention may employ anucleotide construct that is not capable of directing, in a transformedplant, the expression of a protein or an RNA.

In addition, it is recognized that methods of the present invention donot depend on the incorporation of the entire nucleotide construct intothe genome. Rather, the methods of the present invention only requirethat the plant or cell thereof is altered as a result of theintroduction of the nucleotide construct into a cell. In one embodimentof the invention, the genome may be altered following the introductionof the nucleotide construct into a cell. For example, the nucleotideconstruct, or any part thereof, may incorporate into the genome of theplant. Alterations to the genome of the present invention include, butare not limited to, additions, deletions, and substitutions ofnucleotides in the genome. While the methods of the present invention donot depend on additions, deletions or substitutions of any particularnumber of nucleotides, it is recognized that such additions, deletionsor substitutions comprise at least one nucleotide.

In certain embodiments the nucleic acid sequences of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The combinations generated can include multiplecopies of any one or more of the polynucleotides of interest. Thepolynucleotides of the present invention may be stacked with any gene orcombination of genes to produce plants with a variety of desired traitcombinations, including but not limited to traits desirable for animalfeed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balancedamino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801;5,885,802 and 5,703,409); barley high lysine (Williamson, et al., (1987)Eur. J. Biochem. 165:99-106 and WO 1998/20122) and high methionineproteins (Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, etal., (1988) Gene 71:359 and Musumura, et al., (1989) Plant Mol. Biol.12:123)); increased digestibility (e.g., modified storage proteins (U.S.patent application Ser. No. 10/053,410, filed Nov. 7, 2001) andthioredoxins (U.S. patent application Ser. No. 10/005,429, filed Dec. 3,2001)), the disclosures of which are herein incorporated by reference.The polynucleotides of the present invention can also be stacked withtraits desirable for insect, disease or herbicide resistance (e.g.,Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5723,756; 5,593,881; Geiser, et al., (1986) Gene48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol. 24:825);fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence anddisease resistance genes (Jones, et al., (1994) Science 266:789; Martin,et al., (1993) Science 262:1432; Mindrinos, et al., (1994) Cell78:1089); acetolactate synthase (ALS) mutants that lead to herbicideresistance such as the S4 and/or Hra mutations; inhibitors of glutaminesynthase such as phosphinothricin or basta (e.g., bar gene) andglyphosate resistance (EPSPS gene)) and traits desirable for processingor process products such as high oil (e.g., U.S. Pat. No. 6,232,529);modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.5,952,544; WO 1994/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)) and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase and acetoacetyl-CoA reductase (Schubert, etal., (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides affecting agronomic traitssuch as male sterility (e.g., see, U.S. Pat. No. 5,583,210), stalkstrength, flowering time or transformation technology traits such ascell cycle regulation or gene targeting (e.g., WO 1999/61619; WO2000/17364; WO 1999/25821), the disclosures of which are hereinincorporated by reference.

These stacked combinations can be created by any method, including butnot limited to cross breeding plants by any conventional or TopCrossmethodology or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences of interest can be driven bythe same promoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of a polynucleotide of interest. This may be accompanied byany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant.

The nucleotide constructs of the invention also encompass nucleotideconstructs that may be employed in methods for altering or mutating agenomic nucleotide sequence in an organism, including, but not limitedto, chimeric vectors, chimeric mutational vectors, chimeric repairvectors, mixed-duplex oligonucleotides, self-complementary chimericoligonucleotides and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use, such as, for example, chimeraplasty, areknown in the art. Chimeraplasty involves the use of such nucleotideconstructs to introduce site-specific changes into the sequence ofgenomic DNA within an organism, e.g., U.S. Pat. Nos. 5,565,350;5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, all of whichare herein incorporated by reference. See also, WO 1998/49350, WO1999/07865, WO 1999/25821, and Beetham, et al., (1999) Proc. Nad. Acad.Sci. USA 96:8774-8778, herein incorporated by reference. The followingexamples are offered by way of illustration and not by way oflimitation. All references cited herein are hereby expresslyincorporated in their entirety herein by reference.

Other embodiments of the invention include the use of VGT1 and itsalternate forms described herein as markers to screen for and identifyplants which may have altered maturity. The invention thus relates togenetic markers for plants with altered maturity. The markers representpolymorphic variants of the non-coding regulatory element VGT1 that areassociated with RAP2.7 regulation and thus provides a method ofgenotyping plants to determine those more likely to have flowering timethat is altered from wildtype.

Thus, the invention relates to genetic markers and methods ofidentifying those markers in plants, whereby the plant is more likely tohave a maturity that is earlier than normal by means of a mutant VGT1which does not down regulate RAP2.7 appropriately.

Any method of identifying the presence or absence of these markers maybe used, including, for example, single-strand conformation polymorphism(SSCP) analysis, base excision sequence scanning (BESS), RFLP analysis,heteroduplex analysis, denaturing gradient gel electrophoresis andtemperature gradient electrophoresis, allelic PCR, ligase chain reactiondirect sequencing, mini sequencing, nucleic acid hybridization,micro-array-type detection of the VGT1 regulatory element.

The following is a general overview of techniques which can be used toassay for the polymorphisms of the invention.

In the present invention, a sample of genetic material is obtained froma plant.

Isolation and Amplification of Nucleic Acid

Samples of genomic DNA are isolated from any convenient source includingany suitable cell or tissue sample with intact interphase nuclei ormetaphase cells. The cells can be obtained from solid tissue as from afresh or preserved plant part or from a tissue sample. The sample cancontain compounds which are not naturally intermixed with the biologicalmaterial such as preservatives, anticoagulants, buffers, fixatives,nutrients, antibiotics, or the like.

Methods for isolation of genomic DNA from these various sources aredescribed in, for example, Kirby, DNA Fingerprinting, An Introduction,W.H. Freeman & Co. New York (1992). Genomic DNA can also be isolatedfrom cultured primary or secondary cell cultures or from transformedcell lines derived from any of the aforementioned tissue samples.

Samples of RNA can also be used. RNA can be isolated from tissues asdescribed in Sambrook, et al., supra. RNA can be total cellular RNA,mRNA, poly A+RNA, or any combination thereof. For best results, the RNAis purified, but can also be unpurified cytoplasmic RNA. RNA can bereverse transcribed to form DNA which is then used as the amplificationtemplate, such that the PCR indirectly amplifies a specific populationof RNA transcripts. See, e.g., Sambrook, supra, Kawasaki, et al.,Chapter 8 in PCR Technology, (1992) supra, and Berg, et al., (1990) Hum.Genet. 85:655-658.

PCR Amplification

The most common means for amplification is polymerase chain reaction(PCR), as described in U.S. Pat. Nos. 4,683,195; 4,683,202 and4,965,188, each of which is hereby incorporated by reference. Tissuesshould be roughly minced using a sterile, disposable scalpel and asterile needle (or two scalpels) in a 5 mm Petri dish. Procedures forremoving paraffin from tissue sections are described in a variety ofspecialized handbooks well known to those skilled in the art.

To amplify a target nucleic acid sequence in a sample by PCR, thesequence must be accessible to the components of the amplificationsystem. Kits for the extraction of high-molecular weight DNA for PCRinclude a Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim,Indianapolis, Ind.), Genomic DNA Isolation System (GIBCO BRL,Gaithersburg, Md.), Elu-Quik DNA Purification Kit (Schleicher & Schuell,Keene, N.H.), DNA Extraction Kit (Stratagene, LaJolla, Calif.), TurboGenIsolation Kit (Invitrogen, San Diego, Calif.), and the like. Use ofthese kits according to the manufacturer's instructions is generallyacceptable for purification of DNA prior to practicing the methods ofthe present invention.

The concentration and purity of the extracted DNA can be determined byspectrophotometric analysis of the absorbance of a diluted aliquot at260 nm and 280 nm. After extraction of the DNA, PCR amplification mayproceed. The first step of each cycle of the PCR involves the separationof the nucleic acid duplex formed by the primer extension. Once thestrands are separated, the next step in PCR involves hybridizing theseparated strands with primers that flank the target sequence. Theprimers are then extended to form complementary copies of the targetstrands. For successful PCR amplification, the primers are designed sothat the position at which each primer hybridizes along a duplexsequence is such that an extension product synthesized from one primer,when separated from the template (complement), serves as a template forthe extension of the other primer. The cycle of denaturation,hybridization and extension is repeated as many times as necessary toobtain the desired amount of amplified nucleic acid.

In a particularly useful embodiment of PCR amplification, strandseparation is achieved by heating the reaction to a sufficiently hightemperature for a sufficient time to cause the denaturation of theduplex but not to cause an irreversible denaturation of the polymerase(see, U.S. Pat. No. 4,965,188, incorporated herein by reference).Typical heat denaturation involves temperatures ranging from about 80°C. to 105° C. for times ranging from seconds to minutes. Strandseparation, however, can be accomplished by any suitable denaturingmethod including physical, chemical, or enzymatic means. Strandseparation may be induced by a helicase, for example, or an enzymecapable of exhibiting helicase activity. For example, the enzyme RecAhas helicase activity in the presence of ATP. The reaction conditionssuitable for strand separation by helicases are known in the art (see,Kuhn Hoffman-Berling, (1978) CSH-Quantitative Biology, 43:63-67 andRadding, (1982) Ann. Rev. Genetics 16:405-436, each of which isincorporated herein by reference).

Template-dependent extension of primers in PCR is catalyzed by apolymerizing agent in the presence of adequate amounts of fourdeoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP and dTTP)in a reaction medium comprised of the appropriate salts, metal cationsand pH buffering systems. Suitable polymerizing agents are enzymes knownto catalyze template-dependent DNA synthesis. In some cases, the targetregions may encode at least a portion of a protein expressed by thecell. In this instance, mRNA may be used for amplification of the targetregion. Alternatively, PCR can be used to generate a cDNA library fromRNA for further amplification, the initial template for primer extensionis RNA. Polymerizing agents suitable for synthesizing a complementary,copy-DNA (cDNA) sequence from the RNA template are reverse transcriptase(RT), such as avian myeloblastosis virus RT, Moloney murine leukemiavirus RT or Thermus thermophilus (Tth) DNA polymerase, a thermostableDNA polymerase with reverse transcriptase activity marketed by PerkinElmer Cetus, Inc. Typically, the genomic RNA template is heat degradedduring the first denaturation step after the initial reversetranscription step leaving only DNA template. Suitable polymerases foruse with a DNA template include, for example, E. coli DNA polymerase Ior its Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taqpolymerase, a heat-stable DNA polymerase isolated from Thermus aquaticusand commercially available from Perkin Elmer Cetus, Inc. The latterenzyme is widely used in the amplification and sequencing of nucleicacids. The reaction conditions for using Taq polymerase are known in theart and are described in Gelfand, (1989) PCR Technology, supra.

Allele Specific PCR

Allele-specific PCR differentiates between target regions differing inthe presence of absence of a variation or polymorphism. PCRamplification primers are chosen which bind only to certain alleles ofthe target sequence. This method is described by Gibbs, Nucleic AcidRes. 17:12427-2448 (1989).

Allele Specific Oligonucleotide Screening Methods

Further diagnostic screening methods employ the allele-specificoligonucleotide (ASO) screening methods, as described by Saiki et al.,Nature 324:163-166 (1986). Oligonucleotides with one or more base pairmismatches are generated for any particular allele. ASO screeningmethods detect mismatches between variant target genomic or PCRamplified DNA and non-mutant oligonucleotides, showing decreased bindingof the oligonucleotide relative to a mutant oligonucleotide.Oligonucleotide probes can be designed so that under low stringency,they will bind to both polymorphic forms of the allele, but at highstringency, bind to the allele to which they correspond. Alternatively,stringency conditions can be devised in which an essentially binaryresponse is obtained, i.e., an ASO corresponding to a variant form ofthe target gene will hybridize to that allele, and not to the wild-typeallele.

Ligase Mediated Allele Detection Method

Target regions of a test subject's DNA can be compared with targetregions in unaffected and affected family members by ligase-mediatedallele detection. See Landegren et al., Science 241:107-1080 (1988).Ligase may also be used to detect point mutations in the ligationamplification reaction described in Wu et al., Genomics 4:560-569(1989). The ligation amplification reaction (LAR) utilizes amplificationof specific DNA sequence using sequential rounds of template dependentligation as described in Wu, supra, and Barany, Proc. Nat. Acad. Sci.88:189-193 (1990).

Denaturing Gradient Gel Electrophoresis

Amplification products generated using the polymerase chain reaction canbe analyzed by the use of denaturing gradient gel electrophoresis.Different alleles can be identified based on the differentsequence-dependent melting properties and electrophoretic migration ofDNA in solution. DNA molecules melt in segments, termed melting domains,under conditions of increased temperature or denaturation. Each meltingdomain melts cooperatively at a distinct, base-specific meltingtemperature (T_(m)). Melting domains are at least 20 base pairs inlength, and may be up to several hundred base pairs in length.

Differentiation between alleles based on sequence specific meltingdomain differences can be assessed using polyacrylamide gelelectrophoresis, as described in Chapter 7 of Erlich, ed., PCRTechnology, “Principles and Applications for DNA Amplification”, W.H.Freeman and Co., New York (1992), the contents of which are herebyincorporated by reference.

Generally, a target region to be analyzed by denaturing gradient gelelectrophoresis is amplified using PCR primers flanking the targetregion. The amplified PCR product is applied to a polyacrylamide gelwith a linear denaturing gradient as described in Myers et al., Meth.Enzymol. 155:501-527 (1986), and Myers et al., in Genomic Analysis, APractical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95-139(1988), the contents of which are hereby incorporated by reference. Theelectrophoresis system is maintained at a temperature slightly below theTm of the melting domains of the target sequences.

In an alternative method of denaturing gradient gel electrophoresis, thetarget sequences may be initially attached to a stretch of GCnucleotides, termed a GC clamp, as described in Chapter 7 of Erlich,supra. Preferably, at least 80% of the nucleotides in the GC clamp areeither guanine or cytosine. Preferably, the GC clamp is at least 30bases long. This method is particularly suited to target sequences withhigh T_(m)'s.

Generally, the target region is amplified by the polymerase chainreaction as described above. One of the oligonucleotide PCR primerscarries at its 5′ end, the GC clamp region, at least 30 bases of the GCrich sequence, which is incorporated into the 5′ end of the targetregion during amplification. The resulting amplified target region isrun on an electrophoresis gel under denaturing gradient conditions asdescribed above. DNA fragments differing by a single base change willmigrate through the gel to different positions, which may be visualizedby ethidium bromide staining.

Temperature Gradient Gel Electrophoresis

Temperature gradient gel electrophoresis (TGGE) is based on the sameunderlying principles as denaturing gradient gel electrophoresis, exceptthe denaturing gradient is produced by differences in temperatureinstead of differences in the concentration of a chemical denaturant.Standard TGGE utilizes an electrophoresis apparatus with a temperaturegradient running along the electrophoresis path. As samples migratethrough a gel with a uniform concentration of a chemical denaturant,they encounter increasing temperatures. An alternative method of TGGE,temporal temperature gradient gel electrophoresis (TTGE or tTGGE) uses asteadily increasing temperature of the entire electrophoresis gel toachieve the same result. As the samples migrate through the gel thetemperature of the entire gel increases, leading the samples toencounter increasing temperature as they migrate through the gel.Preparation of samples, including PCR amplification with incorporationof a GC clamp, and visualization of products are the same as fordenaturing gradient gel electrophoresis.

Single-Strand Conformation Polymorphism Analysis

Target sequences or alleles at the VGT1 loci can be differentiated usingsingle-strand conformation polymorphism analysis, which identifies basedifferences by alteration in electrophoretic migration ofsingle-stranded PCR products, as described in Orita et al., Proc. Nat.Acad. Sci. 85:2766-2770 (1989). Amplified PCR products can be generatedas described above, and heated or otherwise denatured, to formsingle-stranded amplification products. Single-stranded nucleic acidsmay refold or form secondary structures which are partially dependent onthe base sequence. Thus, electrophoretic mobility of single-strandedamplification products can detect base-sequence difference betweenalleles or target sequences.

Chemical or Enzymatic Cleavage of Mismatches

Differences between target sequences can also be detected bydifferential chemical cleavage of mismatched base pairs, as described inGrompe et al., Am. J. Hum. Genet. 48:212-222 (1991). In another method,differences between target sequences can be detected by enzymaticcleavage of mismatched base pairs, as described in Nelson et al., NatureGenetics 4:11-18 (1993). Briefly, genetic material from a plant and anaffected family member may be used to generate mismatch freeheterohybrid DNA duplexes. As used herein, “heterohybrid” means a DNAduplex strand comprising one strand of DNA from one plant, and a secondDNA strand from another plant, usually a plant differing in thephenotype for the trait of interest. Positive selection forheterohybrids free of mismatches allows determination of smallinsertions, deletions or other polymorphisms that may be associated withVGT1 polymorphisms.

Non-Gel Systems

Other possible techniques include non-gel systems such as TAQMAN™(Perkin Elmer). In this system, oligonucleotide PCR primers are designedthat flank the mutation in question and allow PCR amplification of theregion. A third oligonucleotide probe is then designed to hybridize tothe region containing the base subject to change between differentalleles of the gene. This probe is labeled with fluorescent dyes at boththe 5′ and 3′ ends. These dyes are chosen such that while in thisproximity to each other the fluorescence of one of them is quenched bythe other and cannot be detected. Extension by Taq DNA polymerase fromthe PCR primer positioned 5′ on the template relative to the probe leadsto the cleavage of the dye attached to the 5′ end of the annealed probethrough the 5′ nuclease activity of the Taq DNA polymerase. This removesthe quenching effect allowing detection of the fluorescence from the dyeat the 3′ end of the probe. The discrimination between different DNAsequences arises through the fact that if the hybridization of the probeto the template molecule is not complete, i.e., there is a mismatch ofsome form, the cleavage of the dye does not take place. Thus, only ifthe nucleotide sequence of the oligonucleotide probe is completelycomplimentary to the template molecule to which it is bound willquenching be removed. A reaction mix can contain two different probesequences each designed against different alleles that might be presentthus allowing the detection of both alleles in one reaction.

Yet another technique includes an Invader Assay, which includesisothermic amplification that relies on a catalytic release offluorescence. See Third Wave Technology at world wide web at twt.com.

Non-PCR Based DNA Screening

Hybridization probes are generally oligonucleotides which bind throughcomplementary base pairing to all or part of a target nucleic acid.Probes typically bind target sequences lacking complete complementaritywith the probe sequence depending on the stringency of the hybridizationconditions. The probes are preferably labeled directly or indirectly,such that by assaying for the presence or absence of the probe, one candetect the presence or absence of the target sequence. Direct labelingmethods include radioisotope labeling, such as with P³² or S³⁵. Indirectlabeling methods include fluorescent tags, biotin complexes which may bebound to avidin or streptavidin, or peptide or protein tags. Visualdetection methods include photoluminescents, Texas red, rhodamine andits derivatives, red leuco dye and 3,3′,5,5′-tetramethylbenzidine (TMB),fluorescein, and its derivatives, dansyl, umbelliferone and the like orwith horse radish peroxidase, alkaline phosphatase and the like.

Hybridization probes include any nucleotide sequence capable ofhybridizing to the chromosome where VGT1 resides, and thus defining agenetic marker linked to VGT1, including a restriction fragment lengthpolymorphism, a hypervariable region, repetitive element, or a variablenumber tandem repeat. Further suitable hybridization probes include exonfragments or portions of cDNAs or genes known to map to the relevantregion of the chromosome.

Preferred tandem repeat hybridization probes for use according to thepresent invention are those that recognize a small number of fragmentsat a specific locus at high stringency hybridization conditions, or thatrecognize a larger number of fragments at that locus when the stringencyconditions are lowered.

One or more additional restriction enzymes and/or probes and/or primerscan be used. Additional enzymes, constructed probes, and primers can bedetermined by routine experimentation by those of ordinary skill in theart and are intended to be within the scope of the invention.

Although the methods described herein may be in terms of the use of asingle restriction enzyme and a single set of primers, the methods arenot so limited. One or more additional restriction enzymes and/or probesand/or primers can be used, if desired. Indeed, in some situations itmay be preferable to use combinations of markers giving specifichaplotypes. Additional enzymes, constructed probes and primers can bedetermined through routine experimentation, combined with the teachingsprovided and incorporated herein.

The sequences surrounding the polymorphism will facilitate thedevelopment of alternate PCR tests in which a primer of about 4-30contiguous bases taken from the sequence immediately adjacent to thepolymorphism is used in connection with a polymerase chain reaction togreatly amplify the region before treatment with the desired restrictionenzyme. The primers need not be the exact complement; substantiallyequivalent sequences are acceptable. The design of primers foramplification by PCR is known to those of skill in the art and isdiscussed in detail in Ausubel (ed.), Short Protocols in MolecularBiology, 4th Edition, John Wiley and Sons (1999).

The following is a brief description of primer design.

Primer Design Strategy

Increased use of polymerase chain reaction (PCR) methods has stimulatedthe development of many programs to aid in the design or selection ofoligonucleotides used as primers for PCR. Four examples of such programsthat are freely available via the Internet are: PRIMER by Mark Daly andSteve Lincoln of the Whitehead Institute (UNIX, VMS, DOS, andMacintosh), Oligonucleotide Selection Program (OSP) by Phil Green andLaDeana Hiller of Washington University in St. Louis (UNIX, VMS, DOS,and Macintosh), PGEN by Yoshi (DOS only), and Amplify by Bill Engels ofthe University of Wisconsin (Macintosh only). Generally these programshelp in the design of PCR primers by searching for bits of knownrepeated-sequence elements and then optimizing the T_(m) by analyzingthe length and GC content of a putative primer. Commercial software isalso available and primer selection procedures are rapidly beingincluded in most general sequence analysis packages.

Sequencing and PCR Primers

Designing oligonucleotides for use as either sequencing or PCR primersrequires selection of an appropriate sequence that specificallyrecognizes the target, and then testing the sequence to eliminate thepossibility that the oligonucleotide will have a stable secondarystructure. Inverted repeats in the sequence can be identified using arepeat-identification or RNA-folding program such as those describedabove. If a possible stem structure is observed, the sequence of theprimer can be shifted a few nucleotides in either direction to minimizethe predicted secondary structure. The sequence of the oligonucleotideshould also be compared with the sequences of both strands of theappropriate vector and insert DNA. Obviously, a sequencing primer shouldonly have a single match to the target DNA. It is also advisable toexclude primers that have only a single mismatch with an undesiredtarget DNA sequence. For PCR primers used to amplify genomic DNA, theprimer sequence should be compared to the sequences in the GenBankdatabase to determine if any significant matches occur. If theoligonucleotide sequence is present in any known DNA sequence or, moreimportantly, in any known repetitive elements, the primer sequenceshould be changed.

The following examples serve to better illustrate the invention and arenot intended to limit the scope of the invention in any way. Allreferences and patents disclosed herein are specifically incorporatedherein in their entirety by reference.

-   Salvi S., Morgante M., Fengler K., Meeley R., Ananiev E., Svitashev    S., Bruggemann E., Niu X., Li B., Tingey S V., Tomes D., Miao G.-H.,    Phillips R L., Tuberosa R. Progress in the positional cloning of    Vgt1, a QTL controlling flowering time in maize (2003). Proceedings    of 57^(th) Corn and Sorghum and 32^(nd) Soybean Seed Research    Conference. Dec. 11-13, 2002, Chicago.-   Phillips R. L., Kim T. S., Kaeppler S. M., Parentoni S. N.,    Shaver D. L., Stucker R. I. and Openshaw S. J. 1992. Genetic    dissection of maturity using RFLPs. Proc. 47th Ann. Corn and Sorghum    Res. Conf. 47:135-150.-   Vladutu, C., McLaughlin J. and Phillips R. L. 1999. Fine Mapping and    Characterization of Linked Quantitative Trait Loci Involved in the    Transition of the Maize Apical Meristem From Vegetative to    Generative Structures. Genetics 153: 993-1007.-   Salvi S., Tuberosa R., Chiapparino E., Maccaferri M., Veillet S.,    van Beuningen L., Isaac P., Edward K. J., Phillips R. L. (2002).    Toward positional cloning of Vgt1, a QTL controlling the transition    from the vegetative to the reproductive phase in maize. Plant Mol    Biol 48:601-613.

EXAMPLES Example 1 RAP2.7 Expression Level is Associated withDifferences in Maturity

It was determined that Rap2.7 gene expression level determines thetransition to flowering in plants and that vgt1 is a cis-element thatregulates RAP2.7 transcription. Two plants with different maturities(N28 and C22-4) were then screened to identify if the RAP2.7 expressionlevels differ between them.

RNA was synthesized from N28 and C22-4 from different tissues and stagesof development, and RAP2.7 expression was measured by RT-PCR.

Tissue Types:

Mature leaves—exposed, blades+sheaths

Immature leaves—in whorl, blades+sheaths

Shoot apical meristems

Roots—whole, including root apical meristems

Stalks—leftovers

Developmental Stages

Sample Number Genotype 1 2 3 4 5 N28 (late) veg veg veg trans rep C22-4(early) veg trans rep rep rep

Gene Expression Assay

First total RNA was prepared from frozen tissue samples. cDNA was thenmade by reverse transcriptase and the PAR2.7 region was amplified usingPCR with agarose gel and ethidium bromide staining. Band fluorescencewas quantified and tubulin was used as an internal control, and tonormalize expression levels.

FIG. 1 shows the levels of RAP2.7 expression at Day: 14 beforetransition, C22-4 on transition at 20 days, and N28 at transition at 27days. Last two dates have 3 samples of each. P values are significantlydifferent at the first two sampling dates, but not at the latest date.There is RAP2.7 signal from meristems, and other tissues. RAP2.7 is arepressor of flowering, and must be below a particular level forflowering to occur.

The results indicate that RAP2.7 is expressed in every tissue type.RAP2.7 expression levels are lower in C22-4 than in N28 before thereproductive transition (mature leaves), and RAP2.7 expression levelsdecrease during development in both C22-4 and N28. Thus, overexpressionof RAP2.7 will delay transition from vegetative state to flowering.

Example 2 Over-Expression of Maize Rap2.7 Under a Moderate-StrengthConstitutive Promoter

The cDNA sequences of RAP2.7 were obtained from a RT-PCR experiment.Specifically, total RNA from C22-4 leaves was isolated and used astemplate in RT-PCR with gene-specific primers. The gene-specific primerswere designed based on RAP2.7 genomic sequences from B73 genotype. Theprimer sequences are sense -ATGCAGTTGGATCTGAACGT (SEQ ID NO: 9) andantisense -GCCATCACCATCCCCGCTGA (SEQ ID NO:10).

The RT-PCR amplified fragment of RAP2.7, including the entire 1371-bpsequence including the ATG start codon and the TAG stop codon, was fusedto the rice actin promoter and pinII terminator to produce an expressioncassette. This expression cassette was then linked to a selectablemarker cassette containing a bar gene driven by CaMV 35S promoter and apinII terminator in FIG. 2.

Transgenic maize plants were produced by transforming Immature GS3 maizeembryos with this expression cassette, using the Agrobacterium-basedtransformation method described as below.

For Agrobacterium-mediated transformation of maize with the expressioncassette comprising the rice actin promoter operably linked to the maizeRAP2.7 gene, the method of Zhao was employed (U.S. Pat. No. 5,981,840,and PCT patent publication WO98/32326; the contents of which are herebyincorporated by reference). While the method below is described for thetransformation of maize plants with the actin promoter—RAP2.7 expressioncassette, those of ordinary skill in the art recognize that this methodcan be used to produce transformed maize plants with any nucleotideconstruct or expression cassette of the invention that comprises apromoter operably linked to maize RAP2.7 gene for expression in a plant.

Briefly, immature embryos were isolated from maize and the embryoscontacted with a suspension of Agrobacterium, where the bacteria arecapable of transferring the ACTIN promoter-RAP2.7 expression cassette(illustrated above) to at least one cell of at least one of the immatureembryos (step 1: the infection step). In this step the immature embryoswere immersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos were co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). The immature embryoswere cultured on solid medium following the infection step. Followingthis co-cultivation period an optional “resting” step was included. Inthis resting step, the embryos were incubated in the presence of atleast one antibiotic known to inhibit the growth of Agrobacteriumwithout the addition of a selective agent for plant transformants (step3: resting step). The immature embryos were cultured on solid mediumwith antibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos were cultured on medium containing a selective agentand growing transformed callus was recovered (step 4: the selectionstep). Preferably, the immature embryos were cultured on solid mediumwith a selective agent resulting in the selective growth of transformedcells. The resulting calli were then regenerated into plants byculturing the calli on solid, selective medium (step 5: the regenerationstep).

The transformation produced 15 events in GS3XGaspe flint, anearly-flowering genotype. Transformation in GS3XHC69, a normal maturitygenotype, was problematic during shoot regeneration process and onlyproduced 5 events. The problem was presumed to be related to thetransgene. Two of the 5 events had delays in flowering time for up to 30days. In GS3XGaspe flint genotype, majority (16 out of 20) of the eventshad various degrees of delay in flowering time, from 16 days to 36 days.In almost all events with delayed flowering, there was significantchange in plant architecture, mostly increase in plant height andinternode length.

Ectopic expression of Rap2.7 with the rice actin promoter resulted inover expression of the gene, and significantly delayed flowering asmeasured by the number of leaves (nodes) produced prior to flowering.According to the invention, vectors for transformation were produced intwo corn genetic backgrounds (GS3 X HC69, and GS3 X GF, gaspe flint)using the Rap2.7 structural gene with the rice actin promoter.

A brief summary of the T0 phenotype from over-expressing RAP2.7 in maizeis shown in the following tables. As a reference, GS3XGaspe plantscoming out of tissue culture get pollinated (exuding silks) within 45-60days, and have 10 or less leaves. Since these are T0 plants, accuratecounts for leaf number and days to flowering are not possible. Theplants that were late in flowering also had substantial increase ininternode elongation, resulting in increase in plant height. Theseplants also had delayed senescence.

Events GS3XHC69 GS3XGaspe Late 2 16 Total 5 20

Days to Pollination GS3XGaspe Events  <60 days 4 61-70 days 5 71-80 days7  >81 days 1 no ear 3

Leaf Number (estimated) GS3XGaspe Events <10 4 10-12 10 13-14 4 >15 2

Example 3 Down-Regulation of Maize Rap2.7 by RNA Interference

Two fragments from the cDNA sequences of RAP2.7 from genotype C22-4 werecloned by PCR to create an inverted repeat as illustrated below.Specifically, a 955-bp fragment starting at 9-bp downstream from ATG wascloned by PCR and designated as ZM-RAP2.7 (TR1) as a truncated form.Another fragment, 499-bp in length starting from the same position, wascloned and designated as ZM-RAP2.7 (IR1) for inverted repeat FIG. 5.

FIG. 5 Illustration of construction of the RAP2.7 gene fragments for RNAinterference vector. TR1 fragment was then ligated to IR1, with IR1 inreverse orientation. The ligated 2-piece fragment was then linked torice actin promoter with actin 5′-UTR and actin intron1 to create a fullexpression cassette. This expression cassette was then linked to aselectable marker cassette containing a bar gene driven by CaMV 35Spromoter and a pinII terminator.

FIG. 6 is an illustration of the vector used in transformation. Noteonly the portion between the right and left T-DNA borders (RB, LB) isshown. Transgenic maize plants were produced by transforming ImmatureGS3 maize embryos with this expression cassette, using theAgrobacterium-based transformation method described for the RAP2.7over-expression study. The transformation produced 20 transgenic eventsfrom GS3XHC69, a genotype with a normal maturity; and 15 events fromGS3XGaspe flint, an early-flowering genotype. Based on preliminaryobservation on TO plants, all 15 events from the GS3XGaspe flintbackground showed no visible change in flowering time. However, 9 out ofthe 20 GS3XHC69 transgenic events had various degrees of early floweringphenotype. The earliest event flowered approximately 2 week earliercompared to other events with the same construct, and to other unrelatedtransgenic plants in the same greenhouse room. All plants had normalplant architecture.

The T0 data shows that if you down regulate RAP2.7 earlier flowering isobserved. The following table shows the next (T1) generation of plants.We can see from this data that the phenotype is heritable and stable andthe initial trend of decreased days to pollination is still observed.

PHP21842 T1 Phenotype Event- Plant Transgene Leaf # Days to Pollination1-1 + 15 58 1-2 + 13 55 1-3 + 15 57 2-1 + 14 57 2-2 + 14 54 2-3 + 13 533-1 + 14 57 3-2 + 13 57 3-3 + 14 59 Control + 19.2 66 average (n = 20)

Example 4 Positional Cloning of Vgt1

Positional cloning was completed after mapping Vgt1 in a 1.3-cM intervalflanked by two AFLP markers (Salvi et al., 2002), in a cross between twonearly isogenic lines, N28 and its early derivative C22-4 (Vladutu etal., 1999). Following BAC library screening and the analysis of therelevant BAC contig, further development of new markers and geneticmapping allowed for the delimiting of Vgt1 within a ca. 2.7-kb region(FIG. 7.)

Sequencing of the relevant BAC clone and of the corresponding DNA regionfrom the parental lines involved in the cross showed that Vgt1 lieswithin an intergenic region, ca. 75 kb upstream of an Ap-2 like gene(Rap2.7) and ca. 10 kb downstream of a RAD51-like gene. The 2.7 kbregion found to be completely associated with Vgt1 is essentially alow-copy region with a number of polymorphisms between N28 and C22-4(one of the polymorphisms is caused by the insertion of a MITEtransposable element in C22-4).

Surprisingly, this sequence does not code for any known protein. It ishypothesized to either be a RNAi element or a regulatory RNA or DNAelement that either directly regulates expression of flowering genessuch as Rap2.7 or specifically targets expression of other genes whichcontrol flowering genes such as Rap2.7.

Example 5 Vgt1 is Associated with Maturity Shift in Inbred Lines

The information gathered with the positional cloning study allowed thetesting of the 2.7 kb region as candidate for controlling flowering timethrough association mapping. Several SNPs and other polymorphismsidentified at and around Vgt1 were screened on a panel of ca. 100 linesrepresentative of cultivated maize germplasm (Remington et al., 2001)and a panel of elite proprietary lines.

Linkage disequilibrium at the Vgt1 region was quickly dissipated overdistance of ca. 1 kb within the panel of 100 lines. Association analysisshowed that among the genes and sequence at and around Vgt1, the onlyDNA region statistically associated with flowering time was a sub-regionof ca. 2 kb within the same 2.7 kb region identified by positionalcloning (FIG. 8.).

This element thus can be used as a sequence-based marker to identifyinbred and hybrids which have altered maturity.

1. An isolated nucleic acid molecule that encodes a polypeptide havingRAP2.7-like activity, said nucleic acid molecule being selected from thegroup consisting of: (a) a nucleic acid molecule comprising the sequenceset forth in SEQ ID NOS: 1, 3, or 4; (b) a nucleic acid moleculecomprising a sequence encoding the amino acid sequence set forth in SEQID NOS: 2; (c) a nucleic acid molecule comprising a sequence having atleast 80% sequence identity to the nucleotide sequence set forth in SEQID NOS: 1, 3, or 4; and (d) a nucleic acid molecule comprising asequence of at least 50 consecutive nucleic acids of any of (a) through(d).
 2. A vector comprising the nucleic acid molecule of claim
 1. 3. Aplant cell having stably incorporated in its genome the nucleic acidmolecule of claim
 1. 4. The plant cell of claim 3, wherein said plantcell is from a monocot plant.
 5. The plant cell of claim 4, wherein saidmonocot plant is maize.
 6. A plant having stably incorporated into itsgenome the nucleic acid molecule of claims
 1. 7. A method for alteringflowering time in a plant comprising: transforming a plant cell with anucleic acid molecule operably linked to a promoter that regulatestranscription of said sequence in a plant cell; wherein said nucleicacid molecule comprises a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence comprising the sequence setforth in SEQ ID NOS: 1, 3, or 4; (b) a nucleotide sequence encoding theamino acid sequence of SEQ ID NO:2; (c) a nucleotide sequence having atleast 80% sequence identity to the sequence of SEQ ID NOS: 1, 3, or 4,wherein said nucleotide sequence encodes a protein which regulatesflowering in plants.
 8. The method of claim 6, wherein said plant is amonocot.
 9. The method of claim 7, wherein said monocot is maize. 10.The method of claim 7 wherein said nucleic acid molecule is a vector forover-expression of RAP2.7.
 11. The method of claim 7 wherein said plantflowers later than a plant which does not have the overexpression vectorincorporated therein.
 12. The method of claim 7 wherein said nucleicacid moleculare is a vector for inhibiting expression of RAP2.7.
 13. Themethod of claim 12 wherein said vector is an RNA interference vector.14. The method of claim 13 wherein said plant flowers earlier than aplant which does not have an RNA interference vector incorporatedtherein.
 15. A method for altering maturity of a plant, said methodcomprising: transforming said plant with a nucleic acid moleculecomprising a heterologous sequence operably linked to a promoter thatinduces transcription of said heterologous sequence in a plant cell; andregenerating stably transformed plants, wherein said heterologoussequence comprises a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence comprising the sequence setforth in SEQ ID NOS: 1, 3 or 4; (b) a nucleotide sequence comprising atleast 50 contiguous nucleotides of the sequence of SEQ ID NOS: 1, 3, or4, wherein said nucleotide sequence encodes a protein floweringregulatory activity; and (c) a nucleotide sequence having at least 80%sequence identity to the sequence of SEQ ID NOS: 1, 3, or
 4. 16. Theplant of claim 18, wherein said promoter is a constitutive promoter. 17.The plant of claim 18, wherein said promoter is a tissue-preferredpromoter.
 18. The plant of claim 18, wherein said promoter is aninducible promoter.
 19. The method of 15, wherein said plant is amonocot.
 20. The method of 16, wherein said monocot is maize.
 21. Themethod of claim 15 wherein said nucleic acid molecule is a vector forover-expression of RAP2.7.
 22. The method of claim 18 wherein said plantmatures earlier than a plant which does not have the overexpressionvector incorporated therein.
 23. The method of claim 15 wherein saidnucleic acid molecule is a vector for inhibiting expression of RAP2.7.24. The method of claim 20 wherein said vector is an RNA interferencevector.
 25. The method of claim 13 wherein said plant matures later thana plant which does not have an RNA interference vector incorporatedtherein.
 26. An isolated polypeptide having flowering regulatoryactivity and selected from the group consisting of: (a) a polypeptidecomprising the amino acid sequence set forth in SEQ ID NOS: 2; (b) apolypeptide encoded by a nucleotide sequence comprising the sequence setforth in SEQ ID NOS: 1, 3, or 4; (c) a polypeptide encoded by anucleotide sequence that has at least 80% sequence identity to thesequence set forth in SEQ ID NOS: 1, 3, or
 4. (d) a polypeptidecomprising an amino acid sequence having at least 90% sequence identityto the sequence set forth in SEQ ID NO:2; and (e) a polypeptidecomprising an amino acid sequence of at least 30 consecutive amino acidsof any of (a) through (d).
 27. A nucleotide construct comprising: anucleic acid molecule encoding an amino acid of claim 23, wherein saidnucleic acid molecule is operably linked to a promoter that drivesexpression in a host cell.
 28. An isolated nucleic acid molecule thatencodes a regulatory element having regulatory activity for floweringtime, said nucleic acid molecule being selected from the groupconsisting of: (a) a nucleic acid molecule comprising the sequence setforth in SEQ ID NOS: 5, 6, 7, or 8; (b) a nucleic acid moleculecomprising a sequence having at least 80% sequence identity to thenucleotide sequence set forth in SEQ ID NOS: 5, 6, 7, or 8; and (c) anucleic acid molecule comprising a sequence of at least 50 consecutivenucleic acids of any of (a) through (c).
 29. A vector comprising thenucleic acid molecule of claim
 28. 30. A plant cell having stablyincorporated in its genome the nucleic acid molecule of claim
 27. 31.The plant cell of claim 30, wherein said plant cell is from a monocotplant.
 32. The plant cell of claim 31, wherein said monocot plant ismaize.
 33. A plant having stably incorporated in its genome the nucleicacid molecule of claim
 27. 34. A method for changing maturity of aplant, said method comprising: stably introducing into the genome of aplant, at least one nucleotide construct comprising a nucleic acidmolecule operably linked to a heterologous promoter that drivestranscription in a plant cell, wherein said nucleic acid moleculeencodes a regulatory element having regulatory activity on floweringtime and is selected from the group consisting of: (a) a nucleic acidmolecule comprising the sequence set forth in SEQ ID NOS: 5, 6, 7, or 8;(b) a nucleic acid molecule comprising a sequence having at least 80%sequence identity to the nucleotide sequence set forth in SEQ ID NOS: 5,6, 7, or 8; and (c) a nucleic acid molecule comprising a sequence of atleast consecutive nucleic acids of any of (a) through (c).
 35. A methodof changing the maturity of a plant comprising, introducing to a plantcell, a VGT1 expression construct, comprising, a VGT1 regulatory elementwhich regulates RAP2.7 expression, operably linked to a promoter.
 36. Amethod of identifying a plant with altered maturity time, comprising:assaying said plant for a VGT1 polymorphism, wherein said polymorphismis identified in FIG. 8 wherein said VGT1 polymorphism is associatedwith a earlier maturity in said plant than the maturity of a plantwithout said VGT1 polymorphism.